The present invention relates to proteins that interact with Human Immunodeficiency Virus (HIV) proteins. More specifically, the present invention relates to complexes of polypeptides or polynucleotides encoding the polypeptides, fragments of the polypeptides, antibodies to the complexes, Selected Interacting Domains (SID®) which are identified due to the protein-protein interactions, methods for screening drugs for agents which modulate the interaction of proteins and pharmaceutical compositions that are capable of modulating the protein-protein interactions.
In another embodiment, the present invention provides a protein-protein interaction map called a PIM® which is available in a report relating to the protein-protein interactions of particles from HIV.
In yet another embodiment, the present invention relates to the identification of additional proteins in the pathway common to the proteins described therein, such as pathways involved in HIV.
Most biological processes involve specific protein-protein interactions. Protein-protein interactions enable two or more proteins to associate. A large number of non-covalent bonds form between the proteins when two protein surfaces are precisely matched. These bonds account for the specificity of recognition. Thus, protein-protein interactions are involved, for example, in the assembly of enzyme subunits, in antibody-antigen recognition, in the formation of biochemical complexes, in the correct folding of proteins, in the metabolism of proteins, in the transport of proteins, in the localization of proteins, in protein turnover, in first translation modifications, in the core structures of viruses and in signal transduction.
General methodologies to identify interacting proteins or to study these interactions have been developed. Among these methods are the two-hybrid system originally developed by Fields and co-workers and described, for example, in U.S. Pat. Nos. 5,283,173; 5,468,614; and 5,667,973, which are hereby incorporated by reference.
The earliest and simplest two-hybrid system, which acted as basis for development of other versions, is an in vivo assay between two specifically constructed proteins. The first protein, known in the art as the “bait protein” is a chimeric protein which binds to a site on DNA upstream of a reporter gene by means of a DNA-binding domain or BD. Commonly, the binding domain is the DNA-binding domain from either Gal4 or native E. coli LexA and the sites placed upstream of the reporter are Gal4 binding sites or LexA operators, respectively.
The second protein is also a chimeric protein known as the “prey” in the art. This second chimeric protein carries an activation domain or AD. This activation domain is typically derived from Gal4, from VP16 or from B42.
Besides the two-hybrid systems, other improved systems have been developed to detected protein-protein interactions. For example, a two-hybrid plus one system was developed that allows the use of two proteins as bait to screen available cDNA libraries to detect a third partner. This method permits the detection between proteins that are part of a larger protein complex such as the RNA polymerase II holoenzyme and the TFIIH or TFIID complexes. Therefore, this method, in general, permits the detection of ternary complex formation as well as inhibitors preventing the interaction between the two previously defined fused proteins.
Another advantage of the two-hybrid plus one system is that it allows or prevents the formation of the transcriptional activator since the third partner can be expressed from a conditional promoter such as the methionine-repressed Met25 promoter which is positively regulated in medium lacking methionine. The presence of the methionine-regulated promoter provides an excellent control to evaluate the activation or inhibition properties of the third partner due to its “on” and “off” switch for the formation of the transcriptional activator. The three-hybrid method is described, for example in Tirode et al., The Journal of Biological Chemistry, 272, No. 37 pp. 22995-22999 (1997) incorporated herein by reference.
Besides the two and two-hybrid plus one systems, yet another variant is that described in Vidal et al, Proc. Natl. Sci. 93 pgs. 10315-10320 called the reverse two- and one-hybrid systems where a collection of molecules can be screened that inhibit a specific protein-protein or protein-DNA interactions, respectively.
A summary of the available methodologies for detecting protein-protein interactions is described in Vidal and Legrain, Nucleic Acids Research Vol. 27, No. 4, pgs. 919-929 (1999); and Legrain and Selig, FEBS Letters 480, pgs. 32-36 (2000), which references are incorporated herein by reference.
However, the above conventionally used approaches and especially the commonly used two-hybrid methods have their drawbacks. For example, it is known in the art that, more often than not, false positives and false negatives exist in the screening method. In fact, a doctrine has been developed in this field for interpreting the results and in common practice an additional technique such as co-immunoprecipitation or gradient sedimentation of the putative interactors from the appropriate cell or tissue type are generally performed. The methods used for interpreting the results are described by Brent and Finley, Jr. in Ann. Rev. Genet., 31 pgs. 663-704 (1997). Thus, the data interpretation is very questionable using the conventional systems.
One method to overcome the difficulties encountered with the methods in the prior art is described in WO99/42612, incorporated herein by reference. This method is similar to the two-hybrid system described in the prior art in that it also uses bait and prey polypeptides. However, the difference with this method is that a step of mating at least one first haploid recombinant yeast cell containing the prey polypeptide to be assayed with a second haploid recombinant yeast cell containing the bait polynucleotide is performed. Of course the person skilled in the art would appreciate that either the first recombinant yeast cell or the second recombinant yeast cell also contains at least one detectable reporter gene that is activated by a polypeptide including a transcriptional activation domain.
The method described in WO99/42612 permits the screening of more prey polynucleotides with a given bait polynucleotide in a single step than in the prior art systems due to the cell to cell mating strategy between haploid yeast cells. Furthermore, this method is more thorough and reproducible, as well as sensitive. Thus, the presence of false negatives and/or false positives is extremely minimal as compared to the conventional prior art methods.
The etiologic agent of AIDS, namely human immunodeficiency virus (HIV), was discovered in 1984 and reliable tests for HIV antibody as well as for the virus itself are currently available. AIDS is caused by HIV, a human retrovirus of the lentivirus group. The four recognized retroviruses belong to two distinct groups; the human T lymphotropic retrovirus (or leukomia) such as HTLV-I and HTLV-II or the human immunodeficiency viruses such as HIV-1 and HIV-2. HTLV-I and HTLV-II are transforming viruses, while HIV-1 and HIV-2 are cytopathic viruses. The most common cause of AIDS throughout the world is HIV-1. HIV-2 is more closely related to some members of a group of simian immunodeficiency viruses and has about 40% sequence identity to HIV-1. HIV-2 has been identified predominantly in western Africa and is believed to be less pathogenic than HIV-1.
HIV-1 has the usual retroviral genes such as env, gal and pol. The gag gene encodes the precursor virion core proteins for the matrix protein (MA), the capsid protein (CA), nucleocapsid protein (NC) and P6. The pol gene encodes the precursor for various virion enzymes such as protease (PR), reverse transcriptase (RT), RNAse H and integrase (IN). The env gene encodes the precursors for the envelope glycoprotein (Env gp) such as surface glycoprotein (gp 120/SU) and transmembrane protein (gp 41/TM).
The transcriptional transactivator (tat) and the regulator of viral expression (rev) genes are each encoded by two overlapping exons and produce small nonvirion proteins which are essential for viral replication. Also, several nonessential genes which are not implicated in viral expression are encoded by HIV-1 such as vif, vpr, vpu and nef.
AIDS is a global epidemic with virtually every country in the world reporting cases. In the United States alone by the mid-1990s, approximately 120,000 cases among adults and adolescents, and approximately 2,000 cases among children less than 13 years old had been reported.
Sexual contact is the major mode of transmission of HIV world wide. The virus can also be transmitted via blood or blood products and infected mothers can transmit HIV to their infants perinatally and as early as the first and second trimester of pregnancy. The virus can also be transmitted from the mother to infant via breast feeding. The prevalence of HIV infection among intravenous drug users is exceptionally high.
The clinical manifestations of HIV infection range from an asymptomatic state to severe disease. The majority of individuals experience no recognizable symptoms upon initial infection but some patients suffer from acute illness about three to six weeks after primary infection. This acute illness is characterized by fever, rigors, arthralgias, myalgias, maculopapulor rash, urticaria, abdominal cramps, diarrhea and aseptic meningitis. Seroconversion generally occurs between 8 to 12 weeks after infection. Neurologic disease is common in HIV-infected individuals, the most common being encephalopathy or AIDS demantia complex.
Currently. AIDS infected patients are treated with HIV anti-proteases in a three cocktail treatment. However, this medication is very costly and although prolongs the life of the AIDS infected individual, does not cure the HIV infection.
Although the development of potent anti-HIV drugs targeting two viral enzymes, such as Reverse transcriptase (RT) and Protease (PR), has allowed HIV-infected people to live longer and to benefit of a higher quality of life, it is clear that these drugs do not cure the HIV infection. Moreover, their prolonged use often results in significant toxicity and in the emergence of drug-resistant viruses. Importantly, the ability of HIV to establish latent reservoirs early in the course of infection ensures the persistence of the virus even in the face of intensive drug therapy and vigorous antiviral immune response. Thus, there is an urgent need for the development of novel anti-HIV therapies to overcome the problems of resistance to the present drugs and to improve treatment efficiency (Greene and Peterlin 2002).
Besides inhibitors of RT and of PR, inhibitors of the third viral enzyme, Integrase (IN) are just entering human clinical trials (Nair 2002). All these inhibitors target the enzymatic activity of these viral enzymes. However, no inhibitors are directed against the interactions between viral proteins and potentially important cellular partners which ensure optimal viral replication in infected cells. HIV having evolved an extraordinary efficient capacity to exploit the cell's molecular machinery in the course of infection, understanding the dynamic interplay of host cell and virus is essential to the effort to control HIV infection.
This shows that it is still needed to explore all mechanisms of HIV particles and to identify drug targets for AIDS.